PROJECT SUMMARY Iron-deficiency and associated anemia are the most prevalent nutritional disorders worldwide, shared by nearly a third of the human population. These disorders persist even though the diet often contains sufficient iron. Extensive effort focused on discovering how to improve the bioavailability of dietary iron and supplements. However, little is known about how the complex microbiota within the digestive tract, which take up and process host dietary iron for their own needs, influence the efficiency with which iron is absorbed by the host. At the same time, unmetabolized heme iron from red meat diets that remains in the colon has been associated with the development of colon cancer, with microbial activity postulated to play a key role. Understanding how microbial iron metabolism promotes or ameliorates human disease represents a critical knowledge gap. The long-term goal of this work is to treat iron deficiency and associated iron-related disorders by probiotic therapies and/or engineered manipulation of the gut microbiome, supporting the mission of the NIDDK. In this proposal, we hypothesize that, because bacteria absorb iron for their own needs, the microbiome acts as a sink for dietary iron, increasing its residence time in the gut, altering its chemical speciation, and minimizing fecal excretion. Specifically, we hypothesize that the limited O2 environment of the digestive tract and the varying ability of the resident microbiota to use it are key variables controlling the rate of uptake and ultimate fate of iron. These hypotheses will be tested via two aims. In Aim 1, we will examine the uptake, heme/non-heme source dependence, and fate of iron supplied to representative gut bacteria and consortia under controlled O2 atmospheres. These ex situ experiments will allow us to predict how bacteria might interact with dietary iron inside the digestive tract of an animal host. In Aim 2, we will determine how the presence of specific microbes and microbial communities influence the bioavailability and metabolic fate of iron in germ free mice compared to mice with defined microbiomes, including the monocultures and consortia from Aim 1 and complex murine and human microbiomes. Successful completion of this work will provide a first-ever assessment of the bacterial contribution to iron metabolism in an animal host. It will establish working experimental systems and collaborative relationships that will allow for increasingly sophisticated future investigations.